Journal of Reproduction & Infertility

Journal of Reproduction & Infertility

Evaluation of Sperm DNA Fragmentation Using Halosperm Technique after the Freezing–Thawing Process in Men: A Study on the Validation of the SCD Protocol

Authors
Chaymae Rochdi 1
Larbi Allai 2
Ibtissam Bellajdel 3
Hafsa Taheri 1
Hanane Saadi 1
Ahmed Mimouni 1
Mohammed Choukri 1
1 Maternal-Child and Mental Health Research Laboratory, Faculty of Medicine and Pharmacy, Mohammed First University, Oujda, Morocco; Medically Assisted Procreation Unit, Central Laboratory, Mohammed VI University Hospital Center, Ou
2 Laboratory of Sustainable Agriculture Management, Higher School of Technology Sidi Bennour, Chouaib Doukkali University, El Jadida, Morocco
3 Medically Assisted Procreation Unit, Central Laboratory, Mohammed VI University Hospital Center, Oujda, Morocco; Obstetrics Gynecology Service, Mohammed VI University Hospital Center, Oujda, Morocco
10.18502/jri.v25i1.15194
Abstract
Background: DNA fragmentation index (DFI) enhances routine semen analysis by providing valuable insights into male reproductive potential. Utilizing Halosperm test, a sperm chromatin dispersion (SCD) assay based on induced condensation. The purpose of this study was to assess sperm DNA damage both before and after freezing. By following the specified kit instructions, an attempt was made to validate the SCD test protocol, with a particular emphasis on the implications of sperm freezing on its DNA integrity. Methods: In total, 380 fresh human semen samples from normozoospermic patients were frozen at -20°C for 10 days, using SCD cryopreservation reagent. Routine semen analysis and DNA fragmentation index (DFI) were determined for each sample before freezing and after thawing. Semen morphology and sperm DFI were compared before and after freezing/thawing process. Results: There was a significant decrease in sperm normal morphology after thawing (9.31±2.42% vs. 7.1±1.53%, p<0.05, respectively). The sperm head, midpiece, and tail defect rate increased after freezing at -20°C. Moreover, DFI was significantly higher after thawing compared to before freezing (20.71±1.61% before freezing vs. 29.1±0.21% after thawing with p<0.001). Conclusion: Cryoconservation of semen samples at -20°C for 10 days using SCD cryopreservation reagent seems to damage sperm morphology, resulting in a reduction in sperm DNA integrity. The measurement of DFI on a fresh sample remains the most reliable technique for obtaining accurate results.
Keywords
Cryopreservation
DNA Fragmentation
Freezing
Halosperm test
Spermatozoa
Thawing
Björndahl L, Brown JK. The sixth edition of the WHO laboratory manual for the examination and processing of human semen: ensuring quality and standardization in the basic examination of human ejaculates. Fertil Steril. 2022;117(2):246-51.
Sharma A, Minhas S, Dhillo WS, Jayasena CN. Male infertility due to testicular disorders. J Clinl Endocrinol Metab. 2021;106(2):e442-e59.
Bosch E, De Vos M, Humaidan P. The future of cryopreservation in assisted reproductive technologies. Front Endocrinol (Lausanne). 2020;11:67.
Gilbert K, Nangia AK, Dupree JM, Smith JF, Mehta A. Fertility preservation for men with testicular cancer: is sperm cryopreservation cost effective in the era of assisted reproductive technology? Urol Oncol. 2018;36(3):92.e1-92.e9.
Taylor MJ, Weegman BP, Baicu SC, Giwa SE. New approaches to cryopreservation of cells, tissues, and organs. Transfus Med Hemother. 2019;46(3):197-215.
Alahmar AT, Singh R, Palani A. Sperm DNA fragmentation in reproductive medicine: a review. J Hum Reprod Sci. 2022;15(3):206-18.
Le MT, Nguyen TTT, Nguyen TT, Nguyen VT, Nguyen TTA, Nguyen VQH, et al. Cryopreservation of human spermatozoa by vitrification versus conventional rapid freezing: Effects on motility, viability, morphology and cellular defects. Eur J Obstet Gynecol Reprod Biol. 2019;234:14-20.
Upadhyay VR, Ramesh V, Dewry RK, Kumar G, Raval K, Patoliya P. Implications of cryopreservation on structural and functional attributes of bovine spermatozoa: an overview. Andrologia. 2021;53(8):e14154.
Mohammadi F, Varanloo N, Heydari Nasrabadi M, Vatannejad A, Amjadi FS, Javedani Masroor M, et al. Supplementation of sperm freezing medium with myoinositol improve human sperm parameters and protects it against DNA fragmentation and apoptosis. Cell Tissue Bank. 2019;20(10):77-86.
Lu JC, Jing J, Chen L, Ge YF, Feng RX, Liang YJ, et al. Analysis of human sperm DNA fragmentation index (DFI) related factors: a report of 1010 subfertile men in China. Reprod Biol Endocrinol. 2018;16(1):23.
Panner Selvam MK, Sengupta P, Agarwal A. Sperm DNA fragmentation and male infertility. In: Arafa M, Elbardisi H, Majzoub, A, Agarwal A, editors. Genetics of male infertility. Switzerland: Springer, Cham; 2020. p. 155-72.
Chua SC, Yovich SJ, Hinchliffe PM, Yovich JL. How well do semen analysis parameters correlate with sperm DNA fragmentation? a retrospective study from 2567 semen samples analyzed by the Halosperm test. J Pers Med. 2023;13(3):518.
Le MT, Nguyen TT, Nguyen TT, Van Nguyen T, Nguyen TA, Nguyen QH, et al. Does conventional freezing affect sperm DNA fragmentation? Clin Exp Reprod Med. 2019;46(2):67-75.
Rochdi Ch, Bellzjdel I, El Moudane A, El Assri S, Mamri S, Taheri H, et al. The effects of varicocelectomy on sperm DNA fragmentation and conventional semen parameters in men with severe oligoasthenoteratozoospermia. Int J Fertil Steril. 2023. In press.
Zhang F, Li J, Liang Z, Wu J, Li L, Chen C, et al. Sperm DNA fragmentation and male fertility: a retrospective study of 5114 men attending a reproductive center. J Assist Reprod Genet. 2021;38(5):1133-41.
Kim SM, Kim SK, Jee BC, Kim SH. Effect of sperm DNA fragmentation on embryo quality in normal responder women in in vitro fertilization and intracytoplasmic sperm injection. Yonsei Med J. 2019;60(5):461-6.
Lusignan MF, Li X, Herrero B, Delbes G, Chan PTK. Effects of different cryopreservation methods on DNA integrity and sperm chromatin quality in men. Andrology. 2018;6(6):829-35.
Ozimic S, Ban-Frangez H, Stimpfel M. Sperm cryopreservation today: approaches, efficiency, and pitfalls. Curr Issues Mol Biol. 2023;45(6):4716-34.
Raju R, Bryant SJ, Wilkinson BL, Bryant G. The need for novel cryoprotectants and cryopreservation protocols: Insights into the importance of biophysical investigation and cell permeability. Biochim Biophys Acta Gen Subj. 2021;1865(1):129749.
Thomson LK, Fleming SD, Barone K, Zieschang JA, Clark AM. The effect of repeated freezing and thawing on human sperm DNA fragmentation. Fertil Steril. 2010;93(4):1147-56.
Lehti MS, Sironen A. Formation and function of sperm tail structures in association with sperm motility defects. Biol Reprod. 2017;97(4):522-36.
Zandiyeh S, Shahverdi A, Ebrahimi B, Sabbaghian M. A novel approach for human sperm cryopreservation with AFPIII. Reprod Biol. 2020;20(2):169-74.
Spano M, Cordelli E, Leter G, Lombardo F, Lenzi A, Gandini L. Nuclear chromatin variations in human spermatozoa undergoing swim-up and cryopreservation evaluated by the flow cytometric sperm chromatin structure assay. Mol Hum Reprod. 1999;5(1):29-37.
Isachenko E, Isachenko V, Katkov II, Rahimi G, Schondorf T, Mallmann P, et al. DNA integrity and motility of human spermatozoa after standard slow freezing versus cryoprotectant‐free vitrification. Hum Reprod. 2004;19(4):932-9.
Duru NK, Morshedi M, Schuffner A, Oehninger S. Cryopreservation-thawing of fractionated human spermatozoa and plasma membrane translocation of phosphatidylserine. Fertil Steril. 2001;75(2):263-8.
Moghadam MT, Fard YA, Saki G, Nikbakht R. Effect of vitamin D on apoptotic marker, reactive oxygen species and human sperm parameters during the process of cryopreservation. Iran J Basic Med Sci. 2019;22(9):1036-43.
Thu NTM, Trang NT. Comparison of the sperm testing kit with the Halosperm testing kit in an analysis of sperm DNA fragmentation. Vietnam J Sci Technol Eng. 2020;62(2):71-6.
Cankut S, Dinc T, Cincik M, Ozturk G, Selam B. Evaluation of sperm DNA fragmentation via halosperm technique and TUNEL assay before and after cryopreservation. Reprod Sci. 2019;26(12):1575-81.
Journal of Reproduction & Infertility
Volume 25, Issue 1
Winter 2024
Pages 12-19